Dual Pore - Control and Sensor Device

ABSTRACT

Two-pore devices and method for sequencing are described. A two-pore device can include first chamber, a second chamber, and a third chamber, wherein the first chamber is in communication with the second chamber through a first nanopore, and wherein the second chamber is in communication with the third chamber through a second nanopore. The device can also include sensing circuitry for measuring electrical signals associated with a target at a nanopore, and a control circuitry for controlling motion of the target at a nanopore. The device can include and/or switch between sensing and control modes for each of the first nanopore and the second nanopore. Sequencing methods can implement a two-pore device in relation to translocation of a target through one or more nanopores, switching between sensing and control modes as appropriate, and measuring aspects of the target using in sensing modes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/009,007 filed on Jun. 14, 2018, allowed, which claims the benefit ofU.S. Provisional Application No. 62/523,228 filed on Jun. 21, 2017, bothof which are incorporated by reference in their entirety.

BACKGROUND

A nanopore is a nano-scale conduit that forms naturally as a proteinchannel in a lipid membrane (a biological pore), or is engineered bydrilling or etching the opening in a solid-state substrate (asolid-state pore). When such a nanopore is incorporated into ananodevice comprising two chambers that are separated by the nanopore, asensing device, such as a patch clamp or voltage clamp system, can beused to apply a trans-membrane voltage and measure ionic current throughthe pore.

Nanopores offer great promise for inexpensive whole genome DNAsequencing. Two obstacles to nanopore sequencing: (1) the lack ofsensitivity sufficient to accurately determine the identity of eachnucleotide in a nucleic acid for de novo sequencing (the lack ofsingle-nucleotide sensitivity), and (2) the ability to regulate andcontrol the delivery rate of each nucleotide unit through the nanoporeduring sensing. These two obstacles are often inter-related as theinability to regulate delivery rates is one of the underlying problemsthat can be associated with the lack of single-nucleotide sensitivity.Stated another way, if the DNA is traversing past the sensor toorapidly, then the sensor's function can be compromised. There is noexisting method for addressing obstacle 2 that does not involve the useof enzymes or optics, both of which work only in specialized nanoporetechniques and which incur higher complexity and cost compared toelectrical methods.

BRIEF DESCRIPTION OF DRAWINGS

The disclosed embodiments have advantages and features that will be morereadily apparent from the detailed description, the appended claims, andthe accompanying figures (or drawings). A brief introduction of thefigures is below.

FIG. 1 depicts an example nanopore device with two nanopores, inaccordance with one embodiment.

FIG. 2A-B each depicts example circuitry incorporating the two nanoporesof an example nanopore device, in accordance with two embodiments.

FIG. 3 depicts an example two nanopore device with a sensing circuitryand a control circuitry option for each pore, and a switch between thetwo options for each pore, in accordance with one embodiment.

FIG. 4A depicts an example two nanopore device in a first configuration,in accordance with one embodiment.

FIG. 4B depicts an example two nanopore device in a secondconfiguration, in accordance with one embodiment.

FIG. 5 depicts a flow process for sequencing a molecule such as apolynucleotide, in accordance with an embodiment.

DETAILED DESCRIPTION Overview

The present disclosure describes a two-nanopore device in which each ofthe two nanopores are incorporated within a switchable two circuitoption. A first circuitry that incorporates a nanopore, hereafterreferred to as the sensor circuitry, comprises a sensing voltage clampor patch clamp amplifier circuit. When the first circuitry thatincorporates a nanopore is used, the nanopore serves as an “ioniccurrent sensing” nanopore. The second circuitry, hereafter referred toas the control circuitry, comprises customized circuitry that controlsthe magnitude and direction of the field forces across a nanoporeincorporated within the second circuitry. In various embodiments, thecontrol circuitry comprises a phase-locked loop (PLL) or some otherperiodic voltage-control waveform. The control circuitry also has accessto information from the first circuitry (e.g., a measured current) thatcan be used for feedback voltage-control. In this configuration, asensing circuitry is applied to a first nanopore while a controlcircuitry, which is designed for optimal trans-pore voltage-control, isapplied to a second nanopore. Switching between the two circuit typescan be done at any time. In other words, a sensor circuitry can beapplied to the second nanopore while a control circuitry is applied tothe first nanopore. Generally, a control circuitry at one nanopore isused to affect motion of a molecule through the other nanopore, therebyenabling multiple re-readings of the molecule using the sensingcircuitry of the opposite nanopore. In various embodiments, thecombination of the control circuitry and sensing circuitry operated attwo different nanopores can be used to address obstacle 2 describedabove, by slowing the molecule as it translocates through a nanoporeduring controlled delivery and sensing.

An example two nanopore device can be used to capture individualmolecules into two nanopores at one time, and using the sensing circuitto measure the translocation of the molecule through one nanopore. Suchembodiments describing a two-pore device can comprise: a first membranelayer comprising a first nanopore fluidically connecting a first chamberwith a second chamber; and a second membrane layer comprising a secondnanopore fluidically connecting the second chamber to a third chamber,wherein the first nanopore is connected within a sensor circuitry thatapplies a constant voltage across the first nanopore and measurescurrent through the first nanopore, and wherein the second nanopore isconnected within a control circuitry that applies a dynamic voltageacross the second nanopore. In an alternative embodiment, the first poreis connected to a control circuitry and the second pore is connected toa sensing circuitry.

Referring to the first circuitry of each nanopore, the circuitryincorporated can be one of a patch clamp or voltage clamp amplifier. TheTIA of the first circuitry provides a constant field force across thenanopore when the voltage is set constant, with the current measuredthrough the nanopore acting as the sensing signal that detects thepresence and passage of a molecule such as a DNA, RNA, proteins, and anycombination of these molecules (macro-molecules). In this respect, theTIA (patch clamp, voltage clamp) is an example of a “sensing circuit”circuit used in nanopore assays. The voltage is set constant duringsensing, and thus provides no direct control over any passing moleculein the nanopore, but applies a field force that acts on the moleculebefore, during, and after nanopore transit in the range of field-forceinfluence. In various embodiments, the patch clamp is designed foroptimal sensing, not as a voltage actuation mechanism.

Referring to the second circuitry of each nanopore, the controlcircuitry option, which is optimized for DNA motion control, can beimplemented at each nanopore and can use the measurement from the firstcircuit (e.g., measured current) as a feedback signal for feedbackmotion control of the captured molecule. In various embodiments, thevoltage applied by the control circuitry is an oscillatory voltagesignal that is dependent on the feedback signal from the first circuit.For example, the voltage applied by the control circuitry can bemodulated, when desired, as a function of feedback data gathered by thesensor circuitry. Data includes frequency, amplitude, phase, eventduration, quantity, and other comparative relations pertaining to atranslocation event or sequence of translocation events, or patternswithin translocation events (e.g., sequence-specific signatures thatregister as changes in signal depth within the event). As an example, invarious embodiments, the control circuitry applies the dynamic voltageusing a direct current-biased alternating current signal source. Thedynamic voltage can be applied by the control circuitry with a widefrequency range, potentially between 0.001 Hz and 100 MHz and a varyingamplitude range between 0.001V and 10 V. In other embodiments, thevoltages and frequencies applied could be in other ranges.

In various embodiments, the measured current detected by a sensingcircuit is affected by changes in the voltage applied by the controlcircuit, e.g., since voltage changes excite any shared capacitancebetween the pores, including the capacitance of the membranes comprisingeach pore. As such, filters and estimators, including an extended Kalmanfilter implementation, can be designed or co-designed to estimatemolecule-induced changes in the current that are superimposed on thesensing signal.

Also provided herein are methods for determining the sequence of amolecule such as one of a charged polypeptide, polynucleotide,phospholipid, polysaccharide, and polyketide, or another type ofmolecule. The method of sequencing a molecule comprises: a) loading asample comprising a polynucleotide in one of the first or second chamberof the device of any of the above embodiments, wherein the device isconnected to a sensor circuitry, such as a voltage clamp or patch clampsystem, for providing a first voltage across a first nanopore locatedbetween the first chamber and the middle layer, and a second voltageacross a second nanopore located between the middle layer and the secondchamber; (b) setting an initial first voltage and an initial secondvoltage so that the polynucleotide moves through the chambers, therebylocating the polynucleotide across both the first and second nanopores;(c) adjusting the first voltage and the second voltage, wherein the twovoltages are different in magnitude, under controlled conditions, sothat the polynucleotide moves through the first and second nanopores inone direction and in a controlled manner; (d) switching from a sensingcircuitry to a control circuitry at the first pore or the second pore,and employing the control circuitry at said pore for enhanced controlleddelivery of the polynucleotide through the other pore still using thesensing circuitry (the “sensing nanopore”); and (e) identifying eachnucleotide of the polynucleotide that passes through the sensingnanopore.

Example Nanopore Device

In various embodiments, an example nanopore device 100 for employing thetwo-nanopore, one-sensor configuration is a multiple chamber, two-poredevice. With reference to FIG. 1, the example nanopore device 100includes a first chamber 105, a second chamber 110, and a third chamber115. In various embodiments, the first chamber 105 is located within acover 170 that may be composed of an insulating material such as glass.The third chamber 115 is generated on the surface of an insulating layer160 composed of an insulating material such as glass. The chambers areseparated by two membranes (120 a and 120 b) that, in variousembodiments, are composed of a material selected from several options.In a solid-state fabrication process, the membrane material can besilicon nitride, silicon dioxide, aluminum oxide, graphene, anycombinations of these, or any other solid-state material known in theart. An alternative would be a polymer membrane with a biologicalnanopore inserted. Each membrane layer 120 a and 120 b includes aseparate nanopore, hereafter referred to as a first nanopore 125 and asecond nanopore 130. The first nanopore 125 may be a solid-statenanopore, a biological nanopore, or a Field Effect Nanopore Transistor(FENT). The second nanopore 130 may be any of those systems, or an evenlarger micropore (μm scale). The first nanopore 125 is in fluidicconnection with the first chamber 105 and the second nanopore 130 is influidic connection with the third chamber 115.

The depiction of the first, second, and third chambers FIG. 1 is shownas one example and does not indicate that, for instance, the firstchamber is placed above the second or third chamber, or vice versa. Thetwo nanopores 125 and 130 can be arranged in any position so long asthey allow fluid communication between the chambers. Still, in oneaspect, the nanopores are aligned as illustrated in FIG. 1.

In various embodiments, an example nanopore device 100 for employing atwo-nanopore, one-sensor configuration is a two chamber, two-poredevice. As an example, a two chamber, two-pore device can include afirst chamber and second chamber that are each in fluid communicationwith a first 125 and second nanopore 130, respectively. A plurality oflayers can separate the two chambers. For example, the plurality oflayers comprise: a first layer; a second layer; and a conductive middlelayer disposed between the first and second layers. In this two chamber,two-pore device, the first nanopore 125 and second nanopore 130 may beconnected to one another through a channel that is located within theconductive middle layer. A channel refers to any fluid path that enablesfluid flow between the first nanopore 125 and second nanopore 130.

Example Two-Pore, One Sensor

In the present disclosure, a sensor circuitry including a TIA, such as avoltage clamp or patch clamp, is used for applying a constant voltageand detecting ionic changes across a nanopore. Additionally, a controlcircuitry is used at a nanopore to control movement of a molecule. FIG.2A-B each depicts example circuitry incorporating the first 125 andsecond nanopores 130 of an example nanopore device, in accordance withtwo embodiments.

Specifically, FIG. 2A depicts the circuitry of an example multiplechamber, two-pore device 100 (see FIG. 1) that includes a first chamber105, a second chamber 110, and a third chamber 115. In this embodiment,sensing and controlling of a molecule can occur while at least a portionof the molecule resides within the second chamber 110. Additionally,FIG. 2B depicts a two chamber, two-pore device 100 that includes a firstchamber 105, second chamber 110, and a channel 150 located between thefirst nanopore 125 and second nanopore 130. In this embodiment, sensingand controlling of a molecule can occur while at least a portion of themolecule resides within the channel 150.

Although this embodiment depicts two nanopores, the circuitry design canbe applied to more than two nanopores. Additionally, as depicted in theembodiments shown in FIG. 2A and FIG. 2B, the example circuitry includesa sensor circuitry 225 that incorporates the first nanopore 125 and acontrol circuitry 240 that incorporates the second nanopore 130. Inother embodiments, the sensor circuitry 225 may instead incorporate thesecond nanopore 130 whereas the control circuitry 240 incorporates thefirst nanopore 125. In further embodiments, each of the first nanopore125 and second nanopore 130 may be incorporated within a circuitry thatis switchable between a sensor circuitry and a control circuitry.Therefore, sensing and controlling a molecule can be performed at bothnanopores 125 and 130.

Sensor Circuitry

As shown in FIGS. 2A and 2B, the sensor circuitry 225 may be one of avoltage clamp or a patch clamp that 1) applies a static voltage acrossthe second nanopore 130 and 2) captures sensor data as a molecule passesthrough the second nanopore 130.

The nanopore device can include a common voltage for the first 125 andsecond nanopores 130 the sensor circuitry 225. For example, in theembodiment shown in FIG. 2A, the middle chamber 110 of the nanoporedevice can serve as the common voltage for the first 125 and secondnanopores 130. In the embodiment shown in FIG. 2B, the two chamber,two-pore device may include a middle conductive layer 280 that can serveas the common voltage. In various embodiments, the electrical connectionof the middle chamber 110 is achieved through a metallic electrodelocated within the two membrane layers 120 a and 120 b between the twonanopores 125 and 130. In some embodiments, the electrical connection isachieved through a physical connection to a metallic electrode externalto the middle chamber 110. The common voltage potential can refer to areference voltage set by an external system. In some embodiments, thecommon voltage is a common ground for the first nanopore 125 and secondnanopore 130.

The sensor circuitry 225 can be further configured to enable the captureof sensor data corresponding to molecules (e.g., polynucleotide such asDNA) that translocate across the second nanopore 130. In one aspect, thesensor circuitry 225 further includes one or more sensors to capture thesensor data. In one aspect, the sensor includes a pair of electrodesplaced at either side of the second nanopore 130 to measure an ioniccurrent across the second nanopore 130 when a molecule, in particular apolynucleotide, translocates through.

The measured ionic current across the second nanopore 130 is dependenton the geometry of the second nanopore 130. For example, the secondnanopore 130 possesses a resistance R2 within the sensor circuitry 225.The resistance R2 is dependent on the geometry (e.g., diameter) of thesecond nanopore 130. The resistance R2 represents the dynamic poreconductance that is measured by the sensor circuitry 225 to sense thetranslocation of molecules through the second nanopore 130.

In some aspects, the sensor is configured to form a tunnel gap at thesecond nanopore 130 that allows the detection of a molecule whentranslocating through the tunnel gap. When the molecule moves throughthe tunnel gap, the sensor is then able to identify the individualcomponents (e.g., nucleotides) of the molecule. In some embodiments, thesensor is functionalized with reagents that form distinct non-covalentbonds with each nucleotide base. Tunnel sensing with a functionalizedsensor is termed “recognition tunneling.” Using a Scanning TunnelingMicroscope (STM) with recognition tunneling, a DNA base flanked by otherbases in a short DNA oligomer can be identified. Recognition tunnelingcan also provide a “universal reader” designed to hydrogen-bond in aunique orientation to each of the four DNA bases (A, C, G, T) and alsoto the base 5-methyl-cytosine (mC) which is naturally occurring due toepigenetic modifications.

Control Circuitry

The control circuitry controls the motion of a molecule (e.g., DNApolynucleotide, protein, and the like) that is captured into both of thefirst and second nanopores at the same time. Generally, the controlcircuitry applies a directional field force that opposes the field forcearising from the voltage applied by the sensor circuitry at the secondnanopore 130. The control circuitry does not incorporate a voltage clampor patch clamp circuit. Instead, the control circuitry utilizesvoltage-control elements. These voltage-control elements provideperformance for control that surpasses what is possible with a voltageclamp or patch clamp amplifier circuitry (e.g., the sensor circuitry).In particular, such control elements can provide a wide variety ofwaveforms that can be specifically configured to precisely control themotion of a molecule within the two nanopores. Furthermore, the currentmeasurements detected by the sensor circuitry at the second nanopore 130can serve as feedback for the control elements of the control circuitryin real-time.

Referring to the control circuitry 240 in either FIG. 2A or 2B, it caninclude various ways to control both current and voltage. Controlmethods can include, but are not limited to, a voltage-controlledamplifier (VCA), digital control amplifier (DCA), pulse width modulator(PWM), an amplitude control, or a phase loop lock (PLL) workingseparately or in combination. Generally, the control circuitry 240 1)applies a dynamic voltage across the first nanopore 125 and 2) controlsthe movement of a molecule through the second nanopore 130. The controlcircuitry 240 applies a dynamic voltage across the first nanopore 125.The applied dynamic voltage imparts a force upon the molecule thatopposes the force imparted by the static force generated by the sensorcircuitry 225, with an opposing force strength that is less than thestatic force strength for molecule motion toward the sensing pore, orwith an opposing force strength that is greater than the static forcestrength for molecule motion toward the controlling pore. Therefore,varying the dynamic voltage enables the control over the direction ofmotion of the molecule as well as the rate of motion (e.g., velocity) ofthe molecule through the second nanopore 130.

The control circuitry can also be configured to provide an electricfield associated with a direct current (DC) source or an alternatingcurrent (AC) source. In one application, application of a driving force,by way of an AC electric field having an associated frequency can beused to control position, velocity, and/or acceleration of a target at,through, or between one or more of the nanopores of the system.

The control circuitry can receive feedback data that can be used toapply the dynamic voltage. As an example, the feedback data can bedetected by the sensor circuitry 225 (e.g., measured current through ananopore incorporated in the sensor circuitry 225). In one embodiment,the feedback data may be the frequency (e.g., period) in which amolecule repeatedly passes back and forth through the second nanopore130, which is derived from the sensor data captured by the sensorcircuitry 225. Therefore, the applied dynamic voltage can ensure thatthe molecule continues to pass back and forth through the secondnanopore 130 incorporated by the sensing circuitry 225.

To generate the dynamic voltage, the PLL of the control circuitry 240receives the feedback data, which can correspond to a measured currentdetected by the sensing circuitry 225. The measured current can befiltered and compared to a reference signal to generate an error signal(e.g., difference between reference signal and frequency data).Additionally, other filtered versions of the error signal can be used toadjust the control voltage signal in real-time. The first and higherorder derivatives of the error signal, and/or integral(s) of the errorsignal, in addition to a proportional error term, could be used in thefeedback calculation. The reference signal could be known a priori,based on data gathering and learning done in prior experiments, or itcould be generated during the experiment through an adaptive orreal-time learning process or a combination thereof.

In various embodiments, if the molecule is a DNA molecule, an examplereference signal can be attenuation pulses within the DNA signal thatmatch known sequence-specific payloads bound at known sites on a doublestranded DNA (dsDNA) scaffold, with each payload generating a pulse asit passes through a nanopore, relative to the dsDNA signal level withouta payload. In that example, the reference pulse frequency desired couldcorrespond to a known DNA rate through a nanopore. Another referencesignal could be based on a desired rate of change of a measured signalin the feedback data, i.e., to either speed up or slow down thedetection of step changing events within the measured signal, whetherknown a priori to exist or not. Another reference signal is based on adesired phase of frequency data, which can be used in a phase-lock loopcontroller circuit.

The control circuitry 240 may include a feedback controller that isconfigured to stabilize the control voltage signal relative to thatreference signal, in either feedforward or feedback directions. Invarious embodiments, the feedback and feedforward control system couldbe designed and implemented with a sufficiently detailed model of thetotal system, e.g., identified using system identification tools. Thefeedback drives the error to zero (e.g., so that the measured signalwill match a defined reference signal). Even in the presence ofuncertainty, feedforward aids in reference tracking and disturbancerejection, to improve the total system performance. The feedback orfeedforward signal can be designed in either a frequency domain (e.g.,frequency) or a time domain (e.g., period).

In various embodiments, such as those depicted in FIGS. 2A and 2B, thereference signal is processed to determine the phase of the feedbackdata. The output voltage of the phase detector is used to control thevoltage-controlled oscillator (VCO) such that the phase differencebetween the phase of the voltage signal outputted by the VCO and thephase of the reference signal is held constant, thereby making it anegative feedback system. In various embodiments, as depicted in FIG.2A/2B, the feedback loop incorporates a fractional-N synthesizer such asa divide-by-N function. This ensures that the output from the VCO is arational multiple of the reference frequency and can enable comparisonsat specified frequency resolutions.

The voltage output from the PLL is amplified by the voltage-controlledamplifier (VCA) based on an amplitude control. The VCA provides controlof peak voltages applied across the first nanopore 125. The firstnanopore 125 possesses a resistance R1 that is dependent on the geometry(e.g., diameter) of the first nanopore 125. The resistance R1 representsthe dynamic pore conductance that acts as the load for the PLL and VCAoutput.

Altogether, the control circuitry 240 incorporating the first nanopore125 serves as an electromagnetic force circuit. In other words, thevoltage applied across the first nanopore 125 creates an electromagneticfield force which interacts with a molecule located between the membranelayers 120 a and 120 b in the middle chamber 110. The applied forcedirects the molecule in either direction (e.g., towards the firstnanopore 125 and away from the second nanopore 130 or towards the secondnanopore 130 and away from the first nanopore 125), through theselection of the magnitude of the applied voltage across the firstnanopore 125 relative to the magnitude of the applied voltage across thesecond nanopore 130. During control, the voltage polarities are set topull DNA away from the middle chamber between the pores, and the voltagemagnitude of the control circuitry is adjusted relative to the voltageapplied by the sensing circuitry to achieve net motion of DNA in eitherdirection. Therefore, the application of a dynamic voltage that altersthe electromagnetic field force that interacts with the molecule enablesthe repeated back and forth movement of the molecule through the secondnanopore 130.

In various embodiments, the control circuitry 240 employs a periodicvoltage-control mechanism across the first nanopore 125 using a directcurrent (DC)-biased AC signal source. This signal source can tune atleast two parameters that enable the dynamic adjustment of the appliedvoltage and the resulting electric field/force at the first nanopore125:

1) The amplitude (or gain) of the signal source, and2) The period (or frequency) of the signal source.

Other parameters of the input voltage signal such as duty cycle, waveshape (sinusoidal, square, sawtooth), and stop periods may be applied bythe signal source as well. In various embodiments, the signal source maybe a single device such as the AD9102 Digital-to-Analog Converter andWaveform Generator. Such a device can easily produce a wide range (e.g.,frequency range of 0.001 Hz to 100 MHz) of waveforms while controlling:gain, period, duty cycle, and wave shape. In some embodiments, the widefrequency range of waveforms of an input voltage signal can be achievedby employing a variable frequency output phase lock loop (PLL) (or otherclock generator), as depicted in FIG. 2A/2B. The PLL can be placed inseries with a variable gain amplifier. PLLs can be either fixedfrequency or variable with certain ranges (e.g., 8 kHz-250 MHz see: IDT8T49N1012). In various embodiments, multiple PLLs can be included in thecontrol circuitry 240 in series to achieve wider frequency ranges.

Switchable Sensing and Control Circuitry

In various embodiments, the sensor and control circuitry options areavailable at each of the two-pores. FIG. 3 depicts an example twonanopore device with a sensing circuitry 225 and a control circuitry 240option for each nanopore, and a switch 310 between the two options foreach pore, in accordance with one embodiment. In particular, a firstnanopore 125 is incorporated in a first overall circuitry 350A thatincludes a first set of both a sensing circuitry 225A and a controlcircuitry 240A. Additionally, a second nanopore 130 is incorporated in asecond overall circuitry 350B that includes a second set of both asensing circuitry 225B and a control circuitry 240B. Each overallcircuitry 350 includes a switch 310A and 310B that enables switchingbetween a sensing circuitry 225 and control circuitry 240 of eachoverall circuitry 350. In one embodiment, setting each switch 310 canenable sensing across the first nanopore 125 and control at a secondnanopore 130, or vice versa. In various embodiments, the switches 310Aand 310B may be embodied differently than displayed in FIG. 3. Forexample, certain hardware components may be shared between the sensingcircuitry 225 and control circuitry 240 and therefore, each switch 310can be configured such that the function of each circuitry (includingthe requisite hardware components) is appropriately enabled whendesired. These embodiments are described in further detail below in FIG.4A and FIG. 4B.

In these embodiments, each of the first nanopore 125 and second nanopore130 can be incorporated in an overall circuitry 350 with a dual roleof 1) applying dynamic voltages to control movement of molecules and 2)detecting ionic measurements corresponding to translocation eventsacross the nanopore. The switch 310A and 310B of each overall circuitry350 is used to set the role of each overall circuitry 350A and 350B.

As shown in FIG. 3, each sensing circuitry 225 can provide sensor datawhereas each control circuitry 240 receives feedback data. The sensordata from each sensing circuitry 225 can be received and processed by aconfiguration select and signal multiplexer. In various embodiments, themultiplexer can receive and filter the sensor data from each sensingcircuitry 225. For example, the multiplexer filters out noise from eachsensor data. The multiplexer directs the sensor data as feedback data tothe opposite overall circuitry 350. For example, if the sensor data isgenerated by a sensing circuitry 225A of the first overall circuitry350A, then the multiplexer directs the sensor data as feedback data tothe control circuitry 240B of the second overall circuitry 350B.

Reference is now made to FIGS. 4A and 4B, which depict an example twonanopore device in a first and second configuration, respectively, inaccordance with one embodiment. In the first and second configurations,the switches 310 control the connectivity to one sensing circuitry 225and one control circuitry 240. In particular, the closed circuitries(and corresponding sensor data and feedback data) are shown in whiteboxes whereas the unconnected circuitries (e.g., open circuit) and thecorresponding sensor data and feedback data are depicted in shadedboxes.

Referring to FIG. 4A, the first configuration of the two nanopore devicerefers to a first switch 310A connecting the sensing circuitry 225A ofthe first overall circuitry 350A and a second switch 310B connecting thecontrol circuitry 240B of the second overall circuitry 350B. Therefore,the sensing circuitry 225A of the first overall circuitry 350A is usedto detect the translocation of the molecule through the first nanopore125. Additionally, the control circuitry 240B of the second overallcircuitry 350B is used to control the motion of the molecule.

Referring to FIG. 4B, the second configuration of the two nanoporedevice refers to a first switch 310A connecting to the control circuitry240A of the first overall circuitry 350A and a second switch 310Bconnecting the sensing circuitry 225B of the second overall circuitry350B. Therefore, the control circuitry 240A of the first overallcircuitry 350A is used to control the motion of the molecule whereas thesensing circuitry of the second overall circuitry 350B is used to detectthe translocation of the molecule through the second nanopore 130.

In various embodiments, the two nanopore device may be placed inadditional configurations. For example, a third configuration includesconnecting both sensing circuitries 225A and 225B through switches 310Aand 310B, respectively. Therefore, the static voltages applied by thesensing circuitries 225A and 225B across their respective nanopores 125and 130 can be used to draw a molecule through one of the nanopores intothe middle chamber 110 or into the channel 150 located between the twonanopores. In various embodiments, the third configuration of the twonanopore device is implemented after a molecule is initially loaded intoa chamber (e.g., a first chamber 105) of the two nanopore device.

As another example, an additional configuration includes connecting bothcontrol circuitries 240A and 240B through switches 310A and 310B,respectively. This configuration can be utilized in conjunction with anadditional method of sensing molecule translocation through a nanopore.As an example, an optical auxiliary sensor can be implemented tooptically image molecules that may be optically tagged. Therefore, thetwo control circuitries 240A and 240B of the additional configurationcan enable finer control over molecule motion through one or bothnanopores.

Operation of Two-Pore, One Sensor

Generally, a control circuitry 240 and a sensor circuitry 225, as shownin FIG. 2A/2B, or multiple control circuitries 240A/240B and multiplesensor circuitries 225A/225B, as shown in FIG. 3/4A/4B can be employedtogether in a two-pore one sensor device to control the movement of amolecule, such as a DNA segment, for sensing and data collection.Although the subsequent description refers to the two nanopore device ina second configuration state (e.g., sensing circuitry 225B incorporatingthe second nanopore 130 and control circuitry 240A incorporating thefirst nanopore 125), the description can similarly be applied toadditional configuration states (e.g., first configuration state).

For example, in the two-pore device depicted in FIGS. 2A and 2B, thecontrol circuitry 240 applies a dynamically altered voltage across thefirst nanopore 125 that generates a force that directionally opposes theforce generated by the static voltage applied across second nanopore 130by the sensor circuitry 225, with a dynamic magnitude that results incontrolled motion of the molecule in either direction. In particular,the voltage applied by the control circuitry 240 across the firstnanopore 125 can direct the movement of molecules by generating varyingfield force strengths that are in magnitude larger than, equal to, orless than the static force deriving from the voltage applied to thesecond nanopore 130 by the sensor circuitry 225. Therefore, dynamicadjustment of the voltage field force at the first nanopore 125,relative to the static field force at the second nanopore 130, enablescontrol over the net direction of motion of a molecule as well as therate of motion (e.g., velocity) of a molecule situated between bothnanopores 125 and 130 in either the middle chamber 110 or channel 150.

In a related example, in the two-pore device depicted in FIGS. 2A and2B, the control circuitry 240 applies a driving force using an ACelectric field with an associated AC frequency. Control or selection ofthe AC frequency (or another aspect of the AC electric field applyingthe driving force) can be based upon information from the sensorcircuitry 225. For instance, one or more of frequency (e.g., frequencyat which a target passes back and forth through a nanopore), amplitudeof a signal, phase of a signal, event duration (e.g., associated withtarget motion at a pore), quantity of targets, and/or any other suitablefeature of an electrical signal from the sensor circuitry 225 can beused to dynamically adjust aspects of the AC electric field applying thedriving force of the control circuitry 240. Therefore, a driving forcefrom an AC source at one nanopore (e.g., the second nanopore 130) canenable control over the net direction of motion of a molecule as well asthe rate of motion (e.g., velocity) of a molecule situated betweennanopores 125, 130.

In particular, the dynamic voltage applied by the control circuitry 240can have a phase that is shifted in comparison to the phase of thesensor data gathered by the sensor circuitry 225. Therefore, as themolecule passes through the second nanopore 130 in a first direction,the applied dynamic voltage changes such that the force imparted by thedynamic voltage opposes the direction of movement of the molecule. Themolecule then changes directions and passes through the second nanopore130 in a second direction (e.g., opposite of the first direction). Here,the dynamic voltage changes again to oppose the second direction ofmovement of the molecule. This process can be repeated to enable themolecule to pass back and forth through the second nanopore 130 until asufficient measurement of the segment of the molecule is obtained.

By oscillating the less-than or greater-than force at the first nanopore125, relative to the static force at the second nanopore 130, thesegments of the molecule can be sensed many times by the sensorcircuitry 225B by repeatedly passing the molecule through the secondnanopore 130. Doing so can improve the signal of detected ionic changescorresponding to translocation of the molecule across the secondnanopore 130 which is useful for a variety of signal processingpurposes, e.g., to improve sequencing of a molecule such as DNA. Therepeated back and forth passing of the molecule, such as apolynucleotide, through the second nanopore 130 is referred to as“flossing” of the polynucleotide. Specifically, the flossing of the DNAsegment (or a portion of the DNA segment) through the second nanopore130 is in response to applied forces (e.g., electrical forces derivedfrom the applied voltages) and can further include frequency datacorresponding to the rate of translocation of the DNA segment throughthe second nanopore 130. As an example, the frequency data is the periodof a single nucleotide base that begins at an initial position,translocates across the second nanopore 130 in a first direction (e.g.,enter into middle chamber 110 or leave middle chamber 110), translocatesback across the second nanopore 130 in a direction opposite to the firstdirection, and returns to the initial position.

FIG. 5 depicts a flow process for sequencing a molecule such as apolynucleotide, in accordance with an embodiment. Specifically, a samplethat includes the polynucleotide is loaded 505 into a first chamber 105of a nanopore device 100. In some embodiments, the polynucleotide can beloaded into a different chamber (e.g., third chamber 115 as shown inFIG. 2A or second chamber 110 in FIG. 2B). The two nanopore deviceapplies 510 a first voltage across a first nanopore 125 and a secondvoltage across a second nanopore 130. In various embodiments, this canbe accomplished by placing the two nanopore device in a thirdconfiguration state (e.g., both the first nanopore 125 and secondnanopore 130 are incorporated in sensing circuitries 225A and 225B,respectively). Therefore, the first and second voltages are each appliedby a sensing circuitry 225. The polynucleotide translocates 515 from thefirst chamber 105 and through a first nanopore 125. Specifically, thesensor circuitry 225A of the first nanopore 125 can apply a constantvoltage across the first nanopore 125 that generates an electrical forcethat draws the polynucleotide through the first nanopore 125. The sensorcircuitry 225 may be configured to measure changes in ionic currentthrough the first nanopore 125. Therefore, when the polynucleotidetranslocates through the first nanopore 125, the sensor circuitrydetects the translocation event based on a detected change in ioniccurrent. Additionally, the polynucleotide translocates 520 through thesecond nanopore 130 due to the applied voltage by the sensor circuitry225B.

The two nanopore device may switch into a different configuration thatopposes the direction of the movement of the molecule. For example, thetwo nanopore device switches from a third configuration state to a firstconfiguration state or a second configuration state depending on thedirectional movement of the molecule. If the molecule was initiallyloaded into the first chamber 105, then the molecule is directionallyexiting from the first chamber 105 and moving towards the second 110 orthird chamber 115. Therefore, to oppose the movement of the molecule,the two nanopore device can switch from a third configuration into afirst configuration state (e.g., see FIG. 4A). In some embodiments, ifthe molecule was initially loaded into a bottom chamber (e.g., thirdchamber 115 in FIG. 2A or second chamber 110 in FIG. 2B), then themolecule is directionally moving towards the first chamber 105.Therefore, to oppose the movement of the molecule, the two nanoporedevice can switch from a third configuration into a second configurationstate (e.g., see FIG. 4B).

The subsequent description refers to switching the two nanopore deviceto a first configuration state, but can also be applied for a switch tothe second configuration state. In various embodiments, the firstvoltage applied by the circuitry incorporating the first nanopore 125 isadjusted 525. Specifically, the polarity of the sensing circuitry 225Ais set such that it opposes the movement of the molecule. For example,the polarity of sensing circuitry 225A can be reversed from a firstpolarity in the third configuration state to a reverse of the firstpolarity in the first configuration state. Additionally, the secondvoltage applied by the circuitry incorporating the second nanopore 130is also adjusted 530. Specifically, the control circuitry 240B of thesecond overall circuitry 350B applies 320 an adjusted second voltageacross the second nanopore 130 in response to detecting that thepolynucleotide has translocated through the first nanopore 125.Generally, the magnitude of the adjusted second voltage applied by thecontrol circuitry 240 is dynamically changing (e.g., an oscillatingvoltage) such that the electrical force arising due to the adjustedsecond voltage can oppose the static force arising from the adjustedfirst voltage. The second voltage applied by the control circuitry 240has a particular waveform (e.g., varying amplitude/magnitude at aparticular frequency) such that the polynucleotide can similarlyoscillate (e.g., floss) back and forth through the first nanopore 125.As the polynucleotide oscillates, the sensor circuitry 225A can detectionic current changes through the first nanopore 125 that corresponds tothe translocation of nucleotide bases of the polynucleotide. Eachnucleotide base can be read multiple times as the polynucleotide flossesback and forth through the first nanopore 125, thereby enabling the moreaccurate identification 535 of individual nucleotides of thepolynucleotide.

When a single nucleotide base from the polynucleotide has beensufficiently read, a polynucleotide exit state in the applied secondvoltage can be applied by the control circuitry 240B to allow for DNAsegment incrementation. In other words, the second voltage can betemporarily adjusted to allow a subsequent nucleotide base pair totranslocate through the first nanopore 125, at which point the secondvoltage can be resumed to floss the subsequent nucleotide base pair backand forth through the first nanopore 125. The magnitude and frequency ofthe applied second voltage across the second nanopore 130 by the controlcircuitry 240B can be tailored according to frequency informationcorresponding to the ionic current measurements detected by the sensorcircuitry 225A.

In various embodiments, an automated and functional circuitry (e.g.,using state machine or machine learning algorithms in concert withfeedback control) could control both the sensor circuitry 225A and thecontrol circuitry 240B, to continuously monitor the sensed data.Therefore, a section of DNA can be read for optimal performance. Forexample, if the ion current corresponding to a DNA translocation eventthrough the first nanopore 125 is not resolved, then the controlcircuitry 240 can perform a step-wise increase in the applied voltageacross the second nanopore 130. Doing so increases the force opposingthe static force applied by the sensor circuitry 225, thereby slowingthe movement of a DNA segment as it translocates through the firstnanopore 125. This improves the signal to noise ratio for each DNAtranslocation across the first nanopore 125 until the desiredperformance (e.g., signal resolution) is achieved.

Flossing a DNA segment and sensing the segment multiple times using asensing circuitry enables the reduction of signal error to an acceptablelevel. Alignment of signals can be used to achieve consensus sequenceswith acceptable accuracy. In some embodiments, the multiple readscorresponding to multiple DNA translocations can be used to generate aconsensus signal, which can subsequently be used to identify thenucleotide base pair.

Additional Considerations

While embodiments, variations, and examples of two-pore devices andmethods implemented with two-pore devices are described above,alternative embodiments, variations, and examples of the invention(s)described can involve a non-two-pore device. For instance, invariations, second chamber 110 (and variations described thereof) can bea conductive channel of a single pore device, wherein the single poredevice has control circuitry (e.g., by way of gate voltage), sensingcircuitry (e.g., in relation to source-to-drain current flow), with theability to switch between control circuitry and sensing circuitry. Sucha single pore device can be manufactured with a lithography process, adrilling process, or any other suitable process that generates a channelor chamber through layers of material.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

1. (canceled)
 2. A nanopore device comprising: a first chamber, a secondchamber, and a third chamber, wherein the first chamber is incommunication with the second chamber through a first nanopore, andwherein the second chamber is in communication with the third chamberthrough a second nanopore; a sensing circuitry connected to the firstnanopore and configured to apply a constant voltage across the firstnanopore and to measure a sensing current across the first nanopore forsensing a charged polymer translocating across the first nanopore; and acontrol circuitry connected to the second nanopore and configured toapply a dynamic voltage across the second nanopore, the applied dynamicvoltage controlling translocation of the charged polymer across thefirst nanopore and the second nanopore, wherein the applied dynamicvoltage is applied using a direct current-biased alternating currentsignal source.
 3. The nanopore device of claim 2, wherein the sensingcircuitry comprises a transimpedance amplifier.
 4. The nanopore deviceof claim 3, wherein the transimpedance amplifier is one of a patch clampor voltage clamp amplifier.
 5. The nanopore device of claim 2, whereinthe control circuitry comprises a phase lock loop (PLL).
 6. The nanoporedevice of claim 5, wherein the control circuitry is configured togenerate an oscillatory voltage output based on feedback from thesensing circuitry.
 7. The nanopore device of claim 6, wherein a phasedifference between a frequency of the oscillatory voltage output and thefrequency of the sensing current is fixed over time.
 8. The nanoporedevice of claim 6, wherein the oscillatory voltage output is provided toa voltage-controlled amplifier (VCA) that applies the dynamic voltageacross the second nanopore.
 9. The nanopore device of claim 2, whereinthe second chamber is electrically coupled as an electrical return pathfor both the sensing circuitry and the control circuitry of at least oneof the first nanopore or the second nanopore.
 10. A nanopore devicecomprising: a first chamber and a second chamber, wherein the firstchamber is in communication with the second chamber through a firstnanopore and a second nanopore; a sensing circuitry connected to thefirst nanopore and configured to apply a constant voltage across thefirst nanopore and to measure a sensing current across the firstnanopore for sensing a charged polymer translocating across the firstnanopore; and a control circuitry connected to the second nanopore andconfigured to apply a dynamic voltage across the second nanopore, theapplied dynamic voltage controlling translocation of the charged polymeracross the first nanopore and the second nanopore, wherein the applieddynamic voltage is applied using a direct current-biased alternatingcurrent signal source.
 11. The nanopore device of claim 10, wherein thesensing circuitry comprises a transimpedance amplifier.
 12. The nanoporedevice of claim 11, wherein the transimpedance amplifier is one of apatch clamp or voltage clamp amplifier.
 13. The nanopore device of claim10, wherein the control circuitry comprises a phase lock loop (PLL). 14.The nanopore device of claim 10, wherein the device further comprises afirst membrane layer that includes the first nanopore, a second membranelayer that includes the second nanopore, and a conductive middle layerbetween the first membrane and second membrane layer.
 15. A methodcomprising: loading a sample comprising a charged polymer in a firstchamber of a nanopore device, wherein the nanopore device comprises thefirst chamber, a second chamber, a first nanopore, and a secondnanopore, wherein the first chamber and the second chamber are incommunication through the first nanopore and the second nanopore;translocating the charged polymer from the first chamber and through thefirst nanopore by applying a dynamically altered voltage across thesecond nanopore via a control circuitry connected to the secondnanopore; sensing the charged polymer by applying a constant voltageacross the first nanopore via a sensing circuitry connected to the firstnanopore and measuring a sensing current across the first nanopore viathe sensing circuitry.
 16. The method of claim 15, wherein thedynamically altered voltage is determined based on a feedback signalcaptured by the sensing circuitry connected to the first nanopore. 17.The method of claim 16, wherein the feedback signal is the sensingcurrent measured across the first nanopore which is a measure ofmovement of the charged polymer across the first nanopore.
 18. Themethod of claim 17, wherein the measure of movement of the chargedpolymer is a measure of one of a position, a velocity, or anacceleration of the charged polymer.
 19. The method of claim 16, whereinthe feedback signal is designed in either a frequency domain or a timedomain by using one of feedforward or feedback.
 20. The method of claim16, wherein the feedback signal is designed using an estimator and afilter that are designed to estimate molecule-induced changes in thesensing current.
 21. The method of claim 15, wherein the dynamicallyaltered voltage is applied with a frequency range between 0.001 Hz and100 MHz and an amplitude range between 0.001 mV and 10 V.